The ultimate goal of this project is the development of liver cells in tissue culture suitable for studies of in vitro chemical carconogenesis. Available liver cell cultures and liver cell lines retain only a small fraction of mixed function oxidase (MFO) activities of the intact liver. It is these activities which involve cytochrome P450 as the oxidase that activate carcinogens. We propose to determine whether the decrease in MFO activities is due to decreased synthesis or increased turnover of heme, the prosthetic group of cytochrome P450. In addition, the reason for the decreased responsiveness of the cultured cells to chemicals which induce increases of MFO activities in the intact rat, will be investigated. Primary cultures of embryonic chick and adult rat will be used as model systems for study of these phenomena because of the investigators' experience with them. The chick embryo system seems particularly promising because of its known in vivo-like responsiveness to chemical inducers of delta-aminolevulinate synthetase, the rate-limiting step for heme biosynthesis. Sensitive fluorometric and radioactive assays will be used for heme synthesis and MFO activities. Attempts will be made to change the culture conditions in order to correct the lesion(s) that resulted in decreased MFO activities. Once optimal conditions are established for maintenance, and if possible, inducibility of NFO activities, the abilities of cells to produce activated carcinogens will be assessed by the Ames assay for mutagens of Salmonella and the binding of radioactive carcinogen metabolites to cellular macromolecules.